lncRNA OIP5-AS1 attenuates the osteoarthritis progression in IL-1β-stimulated chondrocytes

Abstract In view of the association between long noncoding RNA OIP5-AS1 and osteoarthritis (OA) pathology, the corresponding potential mechanism is worthy of exploration. Primary chondrocytes were identified by morphological observation and immunohistochemical staining of collagen II. The association between OIP5-AS1 and miR-338-3p was analyzed by StarBase and dual-luciferase reporter assay. After the expression of OIP5-AS1 or miR-338-3p in interleukin (IL)-1β-stimulated primary chondrocytes and CHON-001 cells was manipulated, cell viability, proliferation, apoptosis rate, apoptosis-related protein (cleaved caspase-9, Bax) expressions, extracellular matrix (ECM) (matrix metalloproteinase (MMP)-3, MMP-13, aggrecan, and collagen II), PI3K/AKT pathway, and mRNA expressions of inflammatory factors (IL-6 and IL-8), OIP5-AS1, and miR-338-3p were determined by cell counting kit-8, EdU, flow cytometry, Western blot, and quantitative reverse transcription-polymerase chain reaction. As a result, the expression of OIP5-AS1 was downregulated in IL-1β-activated chondrocytes, while miR-338-3p was overexpressed. OIP5-AS1 overexpression reversed the effects of IL-1β on viability, proliferation, apoptosis, ECM degradation, and inflammation in chondrocytes. However, OIP5-AS1 knockdown exhibited opposite effects. Interestingly, the effects of OIP5-AS1 overexpression were partially offset by miR-338-3p overexpression. Furthermore, OIP5-AS1 overexpression blocked the PI3K/AKT pathway by modulating miR-338-3p expression. In sum, OIP5-AS1 promotes viability and proliferation, and inhibits apoptosis and ECM degradation in IL-1β-activated chondrocytes by targeting miR-338-3p through blocking the PI3K/AKT pathway, indicating an attractive strategy for OA treatment.


Introduction
Osteoarthritis (OA) is a type of joint disease, characterized by articular cartilage fibrosis, derangement, ulceration, and loss resulting from various factors [1]. As the aging of the population intensifies globally, OA is more prevalent and turns into a principal health problem in the world [2]. Nevertheless, OA progresses are still undefined, and a lack of efficacious options are provided to control or even reverse the course of OA [2]. Articular cartilage mainly comprises chondrocytes and extracellular matrix (ECM). The chondrocyte matrix is synthesized by chondrocytes that are necessary to maintain the normal structure and the function of the articular cartilage, and also experience many physiological changes in the OA stage, like proliferation and secretory profile [3]. Therefore, identifying the potential mechanism of chondrocyte dysfunction may shed new light on the progression of OA and contribute to discovering a novel effective method for the improvement of OA outcome.

Chondrocyte culture
Primary human chondrocytes were purchased from PromoCell (C-12710, Heidelberg, Germany). Cells were incubated in Dulbecco's Modified Eagle Medium (DMEM) (30-2002, ATCC, Manassas, VA, USA) with 10% fetal bovine serum (FBS; 16140071, Thermo Fisher Scientific) and 1% penicillin-streptomycin solution, and plated in a culture flask at 37°C with 5% CO 2 . Until the convergence rate reached 85%, the chondrocytes were digested and then passaged. The enzyme digestion method was the same as that in primary cells. The morphology of chondrocytes was observed with a microscope. The cells at P2/P3 passage were used in all experiments. Human chondrocyte cell line CHON-001 (CRL-2846) was procured from ATCC. CHON-001 cells were grown in DMEM containing 0.1 mg/mL G-418 (10131027, Gibco, Grand Island, NY) and 10% FBS and were incubated at 37℃ with 5% CO 2 .

Immunohistochemical staining
The expression of collagen II in primary chondrocytes at second, third, or fourth generation was observed using immunohistochemical staining. Following culture, the cells were smeared onto climbing slices, fixed in 4% formaldehyde (P0099, Beyotime, China) for 0.5 h, and permeabilized in 0.2% Triton X-100 (T8200, Solarbio) for 15 min. The slices were later washed with phosphate buffer saline (PBS) and then treated with 3% H 2 O 2 (10011208, Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) for 15 min to repress endogenous peroxidase. After that, the cells were incubated with rabbit anti-Collagen II primary antibody (ab34712, 1/200 dilution, Abcam, Cambridge, MA, USA) overnight at 4°C, followed by treatment with horseradish peroxidase (HRP)-conjugated secondary antibody (ab205718, 1/20,000 dilution, Abcam) for 1 h at room temperature. Diaminobenzidine (DAB, ab103723, 1/100 dilution, Abcam) was used as the chromogen to stain the cells, and then the cells were counterstained with hematoxylin (H8070, Solarbio). The images were captured using a microscope (E800, Nikon, Tokyo, Japan) under ×200 magnification (Scale bars, 20 μm). Positive expression was presented as yellow or brown staining.

EdU assay for detecting cell proliferation
After transfection, the cells were seeded into 96-well plates (4 × 10 3 /well). The cell proliferation was measured by EdU assay according to the manufacturer's protocol. Following 2 h of incubation with 100 μL EdU solution (50 μM, R1053.11, RibBio, Guangzho, China) containing medium, the cells were fixed with 50 μL of 4% paraformaldehyde for 30 min at 37℃. Then, the fixed cells were incubated with 50 μL glycine (2 mg/mL) on a decolorizing shaking bed for 5 min, after the fixative was discarded, followed by washing with PBS and permeabilization with 100 μL Triton X-100 (0.5%). After EdU labeling, the cells were treated with 100 μL of Apollo reaction cocktail, and cell nuclei were stained with Hoechst 33342 (100 μL). Finally, EdU-positive cells were observed under a fluorescence microscope and quantified using Image J.

Quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR)
The total RNAs were extracted using TRIzol (12183555, Thermo Fisher Scientific). PrimeScript™ RT reagent Kit (RR037A, Takara, Dalian, China) was utilized to obtain cDNA from 1 μg of total RNA, with random primers and oligo dT. The miRNA was reversely transcribed into cDNA using PrimeScript™ RT reagent kit, with stem-loop RT primers (listed in Table 1). The cDNA was amplified using TB Green ® Premix Ex Taq™ II (RR820A, Takara) on Thermal Cycler Dice™ Real Time System (TP700, Takara). Relative expression levels of genes were calculated based on the 2 −ΔΔCt method [13]. GAPDH or U6 was the internal control for mRNA or miRNA, respectively. The primer sequences were included in Table 1.

Data analysis
Data were analyzed using GraphPad Prism 8.0 (San Diego, CA, USA) and shown as the mean ± standard deviation (SD, n = 3). The independent sample t-test was utilized to compare the differences between the two groups. Comparisons of multiple groups were completed using analysis of variance. Bonferroni test was employed for Post hoc pairwise comparison. p < 0.05 was regarded as a statistically significant difference.

Identification of primary chondrocytes and downregulation of OIP5-AS1 in OA chondrocytes
As shown in Figure 1a, under the inverted microscope, the primary chondrocytes were observed to be spherical, homogenous in size, and not easy to adhere to the wall. Forty-eight hours after incubation, the cells were oval or spindle like, more adhesive to the wall, and rich in cytoplasm, and cell nuclei presented an elongated morphology. Ninety-five hours after incubation, cells grew rapidly, and spindle-like cells were observed to further proliferate, whereas part of the cells gradually became rectangular or polygonal; moreover, cells began to connect with each other, grown in colonies, adhered to the wall, and covered the bottom of the flask. According to Figure 1b, cells at passage 2 exhibited good coloring; coloring in cells at passage 3 was waning, but still  present; and cells at passage 4 exhibited poor coloring and had a greater variation. Therefore, chondrocytes at passage 2 and 3 were used for the subsequent experiments. In addition, the level of OIP5-AS1 was downregulated in IL-1β-activated chondrocytes (Figure 1c and d, p < 0.001). These findings indicated the downregulation of OIP5-AS1 in IL-1β-stimulated chondrocytes.

OIP5-AS1 modulated ECM degradation and inflammation of IL-1β-activated chondrocytes
ECM synthesized by chondrocytes is necessary to maintain the normal structure and the function of the articular cartilage. In the stage of OA, matrix metalloproteinase (MMP)-3 and MMP-13 are indispensable for cartilage degradation [15]. Aggrecan is also the main structural ingredient of ECM. In addition, MMP-13 is regarded as a primary contributor to cartilage degeneration of OA because it preferentially cleaves collagen II [16]. In line with Figure 6a-c, IL-1β augmented the levels of MMP-3 and MMP-13, while diminishing the levels of aggrecan and collagen II in chondrocytes (p < 0.01). Also, the effects of IL-1β stimulation on these genes were partially mitigated by OIP5-AS1 overexpression (p < 0.01), but were potentiated by OIP5-AS1 knockdown (p < 0.01). In  addition, IL-1β stimulation elevated the levels of IL-6 and IL-8 in chondrocytes (Figure 6d and e, p < 0.001), which was offset by OIP5-AS1 overexpression (Figure 6d and e, p < 0.001), but was further promoted by OIP5-AS1 knockdown (Figure 6d and e, p < 0.01). These data manifested that OIP5-AS1 regulated ECM degradation and inflammation of IL-1β-activated chondrocytes.

miR-338-3p offset the inhibitory effect of OIP5-AS1 on PI3K/AKT signaling activation
In OA progression, the PI3K/AKT pathway plays an important role in cell growth and survival [17]. OIP5-AS1 also regulates PI3K/AKT signaling activation by sponging miRNA [18]. Thus, PI3K/AKT signaling involved in this study was further explored. As shown in Figure 10a, the levels of PI3K and AKT did not change among all groups, whereas miR-338-3p overexpression increased the level of phosphorylation (p)-PI3K and p-AKT and partially counteracted the inhibitory effects of OIP5-AS1 overexpression on the levels of p-PI3K and p-AKT in IL-1β-activated chondrocytes (p < 0.001). In light of Figure 10b-e, miR-338-3p upregulation augmented the levels of p-PI3K/PI3K and p-AKT/AKT, and partially offset the inhibitory effects of OIP5-AS1 overexpression on the levels of p-PI3K/PI3K and p-AKT/AKT in IL-1βactivated chondrocytes (p < 0.001). These data demonstrated that OIP5-AS1 modulated PI3K/AKT signaling of IL-1β-activated chondrocytes by directly targeting miR-338-3p.

Discussion
In the present study, we found that the expression of OIP5-AS1 was downregulated in IL-1β-activated chondrocytes, while miR-338-3p was upregulated. OIP5-AS1 promotes viability and proliferation and inhibits apoptosis and ECM degradation in IL-1β-activated chondrocytes via targeting miR-338-3p by blocking the PI3K/AKT pathway. During the initiation and progression of OA, the organization of the cartilage ECM is disrupted, which in turn damages the mechanical function of the organization. The OA severity is associated with the elevated levels of a series of pro-inflammatory factors [19]. Chondrocytes synthesize or degrade ECM via producing anabolic or catabolic factors [20]. In addition, promoting proliferation and inhibiting apoptosis in chondrocytes have been regarded as the key options to prevent or control the progression of OA [21]. As a consequence, exploring the role of critical OA-associated biomarkers in proliferation, apoptosis, ECM composition, as well as inflammation in chondrocytes might contribute to shedding new light on OA pathology and discovering novel avenues to prevent or control OA development.
The current findings showed that OIP5-AS1 was downregulated in IL-1β-treated chondrocytes, which was consistent with the previous results reported by Zhi et al. [8]. Furthermore, the biological function of OIP5-AS1 in IL-1βstimulated chondrocytes was investigated. It was documented that OIP5-AS1 overexpression offset the effects of IL-1β on inhibiting cell viability, proliferation, and protein levels of aggrecan and collagen II, and promoting the apoptosis rate and protein levels of MMP-3, MMP-13, cleaved caspase-9, Bax, IL-6, and IL-8 in chondrocytes. However, OIP5-AS1 knockdown produced the opposite effects. The functional integrity of ECM, particularly rich in collagen II, is responsible for the function of normal articular cartilage [22]. Aggrecan is also the main structural ingredient of ECM. MMPs are the most important protease enzymes in ECM remodeling [23]. In OA, enzymes, such as MMP-3 and MMP-13, are increased in the joint space and indispensable for cartilage degradation [15]. MMP-13 is regarded as a primary contributor to cartilage degeneration of OA, because it preferentially cleaves collagen II [16]. In this study, OIP5-AS1 overexpression repressed IL-1β-stimulated viability decrease, apoptosis activation, ECM degradation, and inflammation enhancement in chondrocytes. Analogously, a previous report suggested that OIP5-AS1 overexpression drives the viability and suppresses the apoptosis and inflammation of chondrocytes [8].
The existing evidence unveiled that miRNA may have significant diagnostic and therapeutic potential and provide a novel option for OA treatment [24]. As per current findings, miR-338-3p expression is elevated in IL-1β-stimulated chondrocytes, implying miR-338-3p might have an association with OA pathology. Furthermore, we found that miR-338-3p was a direct target of OIP5-AS1 and inversely Figure 9: MiR-338-3p partially offset the regulatory effects of OIP5-AS1 on ECM degradation and inflammation of IL-1β-activated chondrocytes. Chondrocytes were co-transfected with miR-338-3p (M)/ MC and OIP5-AS1-overexpressing vector/empty vector, and exposed to IL-1β stimulation. (a-e) The levels of MMP-3, MMP-13, collagen II, and aggrecan were quantitated using Western blot (a-c), and the levels of inflammatory factors IL-6 and IL-8 were examined using qRT-PCR (d and e). IL-1β, interleukin-1β; MC, mimic control; MMP, matrix metalloproteinase; qRT-PCR, quantitative reverse transcription-polymerase chain reaction. ** p < 0.01, *** p < 0.001 vs IL-1β + vector + MC;^p < 0.01,^^^p < 0.001 vs IL-1β + OIP5-AS1 + MC; ## p < 0.01, ### p < 0.001 vs IL-1β + vector + M. modulated by OIP5-AS1. To clarify the interplay of miR-338-3p and OIP5-AS1 in OA progression, we detected the roles of miR-338-3p and OIP5-AS1 in the viability, apoptosis, and inflammation in IL-1β-stimulated chondrocytes. The data proved that OIP5-AS1 promotes viability and inhibits apoptosis and ECM degradation in IL-1β-activated chondrocytes by directly regulating miR-338-3p. In different biological processes, such as OA progression, the PI3K/AKT pathway plays a critical role in cell growth and survival [17]. It has been reported that AKT blocks pro-caspase-9 and phosphorylates caspase-9, thereby interfering with the apoptotic process [25]. Inactivation of PI3K/AKT/NF-κB pathway alleviates the inflammation and lessens the levels of MMP-3 and MMP-13 in human OA chondrocytes [26]. In the current work, we unraveled that OIP5-AS1 overexpression dampened the PI3K/AKT pathway by modulating miR-338-3p expression. Therefore, OIP5-AS1 may be dependent on blocking the PI3K/AKT pathway, so as to promote viability and inhibit apoptosis and ECM degradation by targeting miR-338-3p in IL-1β-activated chondrocytes. However, a limitation of this study is that since PI3K/AKT pathway is related to almost all cancers, exploring this one alone is far from enough to reveal the molecular mechanisms. Other potential molecular mechanisms still require further investigation.
Collectively, OIP5-AS1 promotes viability and proliferation and inhibits apoptosis and ECM degradation in IL-1β-activated chondrocytes by targeting miR-338-3p through blocking the PI3K/AKT pathway. The present results suggest that OIP5-AS1/miR-338-3p axis plays a regulatory role by blocking the PI3K/AKT signaling, which may be a new method for OA treatment. To better elucidate how OIP5-AS1/miR-338-3p axis ameliorates chondrocyte dysfunction by the PI3K/AKT signaling, PI3K/AKT inhibitors should be utilized in the future study, and more extensive experiments should be carried out in appropriate animal models for verification.